Abstract
BackgroundThere are several methods for quantitating bacterial cells, each with advantages and disadvantages. The most common method is bacterial plating, which has the advantage of allowing live cell assessment through colony forming unit (CFU) counts but is not well suited for high throughput screening (HTS). On the other hand, spectrophotometry is adaptable to HTS applications but does not differentiate between dead and living bacteria and has low sensitivity.ResultsHere, we report a bacterial cell counting method termed Start Growth Time (SGT) that allows rapid and serial quantification of the absolute or relative number of live cells in a bacterial culture in a high throughput manner. We combined the methodology of quantitative polymerase chain reaction (qPCR) calculations with a previously described qualitative method of bacterial growth determination to develop an improved quantitative method. We show that SGT detects only live bacteria and is sensitive enough to differentiate between 40 and 400 cells/mL. SGT is based on the re-growth time required by a growing cell culture to reach a threshold, and the notion that this time is proportional to the number of cells in the initial inoculum. We show several applications of SGT, including assessment of antibiotic effects on cell viability and determination of an antibiotic tolerant subpopulation fraction within a cell population. SGT results do not differ significantly from results obtained by CFU counts.ConclusionSGT is a relatively quick, highly sensitive, reproducible and non-laborious method that can be used in HTS settings to longitudinally assess live cells in bacterial cell cultures.
Highlights
There are several methods for quantitating bacterial cells, each with advantages and disadvantages
The Start Growth Time (SGT) values of various bacterial cell cultures inoculated with various starting concentrations and grown in various conditions (Figure 1A) were determined (Figures 1B and 1D)
A calibration curve was generated by plotting the SGT values against the corresponding starting inoculum values, which were assessed by colony forming unit (CFU) counts on plates (Figures 1C and 1E)
Summary
There are several methods for quantitating bacterial cells, each with advantages and disadvantages. The most common method is bacterial plating, which has the advantage of allowing live cell assessment through colony forming unit (CFU) counts but is not well suited for high throughput screening (HTS). The CFU method has two noteworthy advantages, namely the capacity for counts of any number of bacteria using dilutions, if too many, or concentrations if too few. The most important disadvantage of the CFU method is that clumps of bacteria cells can be miscounted as single colonies; the potential for counting clumps as single units is reason the results are reported as CFU/mL rather than bacteria/mL. Since the CFU method is relatively time-consuming and quite tedious, it has limitations for high throughput screening (HTS) studies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
